Therapeutic CD47 Antibodies

ABSTRACT

Provided are monoclonal antibodies and antigen-binding fragments thereof that bind to, and inhibit the activity of, CD47, as well as monoclonal antibodies and antigen binding fragments thereof that compete with the former for binding to CD47. Also provided are combinations of any of the foregoing. Such antibody compounds are variously effective in 1) treating tissue ischemia and ischemia-reperfusion injury (IRI) in the setting of organ preservation and transplantation, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, and other instances of surgery and/or trauma in which IRI is a component of pathogenesis; 2) in treating autoimmune and inflammatory diseases; and 3) as anti-cancer agents that are toxic to susceptible cancer cells, promoting their phagocytic uptake and clearance, or directly killing such cells.

The present invention relates to antibodies that bind CD47 and their use in treating conditions and disorders, such as ischemia-reperfusion injury (IRI) and cancers, mediated by this receptor.

CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 membrane spanning transmembrane domain, and a cytoplasmic tail that is alternatively spliced. Two ligands bind CD47: thrombospondin-1 (TSP1), and signal inhibitory receptor protein alpha (SIRPalpha). TSP 1 binding to CD47 activates the heterotrimeric G protein Gi, which leads to suppression of intracellular cyclic AMP (cAMP) levels. In addition, the TSP1-CD47 pathway opposes the beneficial effects of the nitric oxide pathway in all vascular cells. The nitric oxide (NO) pathway consists of any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced, or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP). The proper functioning of the NO-cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). In the context of these cellular stresses, the inhibition of the NO-cGMP pathway by the TSP1-CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles. There are many cases in which ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.

SIRPalpha is expressed on hematopoietic cells, including macrophages and dendritic cells. When it engages CD47 on a potential phagocytic target cell, phagocytosis is slowed or prevented. The CD47-SIRPalpha interaction effectively sends a “don't eat me” signal to the phagocyte. Thus, blocking the SIRPalpha-CD47 interaction with a monoclonal antibody in this therapeutic context can provide an effective anti-cancer therapy by promoting the uptake and clearance of cancer cells by the host's immune system. This mechanism is effective in both leukemias and many types of solid tumors.

U.S. Pat. No. 8,236,313 contemplates antibodies that could be useful in the field of ischemia and blood flow to reverse and/or prevent tissue ischemia and related and associated tissue and cell damage, including antibodies that block CD47. No antibodies are actually disclosed.

U.S. Pat. No. 8,101,719 discloses humanized antibodies that bind to CD47 for use in treating hematological disorders. Objects of the invention include humanized anti-CD47 antibodies and small antibody fragments exhibiting reduced antigenicity while retaining their CD47 binding activity and apoptosis-inducing activity. Such antibodies and small fragments are contemplated for use in treating hematological disorders such as various types of leukemias, malignant lymphoma, aplastic anemia, myeodysplastic syndromes, and polycythemia vera. No other properties of these antibodies are disclosed.

PCT International Publication WO 2011/143624 discloses chimeric and humanized anti-CD47 monoclonal antibodies for use as reagents for the diagnosis and immunotherapy of diseases associated with CD47 in humans, particularly in cancer therapy, for example to increase phagocytosis of cancer cells expressing CD47. Preferred antibodies are non-activating, i.e., block ligand binding, but do not signal. Disclosed humanized B6H12 and 5F9 antibodies bound soluble human CD47; B6H12 also bound human CD47 on the surface of human CD47-transfected YB2/0 cells. Humanized B6H12 and 5F9 antibodies enabled phagocytosis of CFSE-labeled HL-60 cells by mouse bone marrow- or peripheral blood-derived macrophages in vitro, respectively. Humanized B6H12 utilized human VH-3-7 and VK3-11 frameworks.

There exists a need for antibodies to human CD47 that selectively block the binding of TSP1 to CD47 to promote the beneficial effects of nitric oxide-cGMP signaling and cAMP signaling in the cardiovascular system in settings in which IRI plays a role in pathogenesis. These situations/diseases include organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resections and reconstructive surgery, reattachment of digits/body parts, skin grafting, and trauma. There is also a need for antibodies that block the binding of SIRPalpha to CD47, thus providing novel anti-cancer therapies. Such antibodies that also have the ability to selectively kill transformed or cancer cells are also expected to provide additional therapeutic benefit.

Antibody compounds of the present invention meet these needs. They bind to epitopes in the extracellular IgV domain of CD47, inhibiting TSP1 and SIRPalpha binding to CD47 and receptor activation, while inducing little or no agonist activity. Certain other antibodies of the present invention also provide a tumor-toxic effect that is specific to activated or transformed cells in addition to promoting tumor cell phagocytic clearance. In view of these properties, antibody compounds of the present invention should be therapeutically useful in treating many forms of IRI and both blood cancers and solid tumors.

In addition, the present antibody compounds possess a number of other desirable properties, including broad reactivity with CD47 of a wide variety of mammalian species, including that of human, mouse, rat, and pig, making these antibodies useful in both human and veterinary medicine. This feature is further advantageous in that it facilitates preclinical studies including, but not limited to, safety and efficacy studies, in a variety of mammalian species, and therefore the development of such antibodies as human and veterinary therapeutics.

Accordingly, the present invention provides:

-   -   [1] A monoclonal antibody, or antigen-binding fragment thereof,         that specifically binds human, rat, mouse, and pig CD47.     -   [2] The monoclonal antibody or antigen-binding fragment thereof         of [1], which is chimeric or humanized.     -   [3] The monoclonal antibody, or antigen-binding fragment         thereof, of [1] or [2], which comprises three light chain         complementarity determining regions (LCDRs 1-3) and three heavy         chain complementarity determining regions (HCDRs 1-3), wherein:         -   LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH             (SEQ ID NO:1) LCDR 2 comprises the amino acid sequence             KVSYRFS (SEQ ID NO:2); and         -   LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID             NO:3);         -   HCDR1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID             NO:4);         -   HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN             (SEQ ID NO:5); and         -   HCDR 3 comprises the amino acid sequence GGYTMDY(SEQ ID             NO:6).     -   [4] The monoclonal antibody, or antigen-binding fragment         thereof, of any one of [1]-[3], which comprises a light chain         variable region (LCVR) and a heavy chain variable region (HCVR),         wherein said LCVR and said HCVR comprise, respectively, amino         acid sequences selected from the group consisting of:         -   SEQ ID NO:7 and SEQ ID NO:57;         -   SEQ ID NO:8 and SEQ ID NO:58;         -   SEQ ID NO:9 and SEQ ID NO:59;         -   SEQ ID NO:10 and SEQ ID NO:60;         -   SEQ ID NO:11 and SEQ ID NO:61;         -   SEQ ID NO:12 and SEQ ID NO:62;         -   SEQ ID NO:13 and SEQ ID NO:63;         -   SEQ ID NO:14 and SEQ ID NO:64;         -   SEQ ID NO:15 and SEQ ID NO:65;         -   SEQ ID NO:16 and SEQ ID NO:66;         -   SEQ ID NO:17 and SEQ ID NO:67;         -   SEQ ID NO:18 and SEQ ID NO:68;         -   SEQ ID NO:19 and SEQ ID NO:69;         -   SEQ ID NO:20 and SEQ ID NO:70;         -   SEQ ID NO:21 and SEQ ID NO:71;         -   SEQ ID NO:22 and SEQ ID NO:72;         -   SEQ ID NO:23 and SEQ ID NO:73;         -   SEQ ID NO:24 and SEQ ID NO:74;         -   SEQ ID NO:25 and SEQ ID NO:75;         -   SEQ ID NO:26 and SEQ ID NO:76;         -   SEQ ID NO:27 and SEQ ID NO:77;         -   SEQ ID NO:28 and SEQ ID NO:78;         -   SEQ ID NO:29 and SEQ ID NO:79;         -   SEQ ID NO:30 and SEQ ID NO:80; and         -   SEQ ID NO:31 and SEQ ID NO:81.     -   [5] A monoclonal antibody, or antigen-binding fragment thereof,         which comprises a light chain variable region (LCVR) and a heavy         chain variable region (HCVR), wherein said LCVR and said HCVR         comprise, respectively, amino acid sequences selected from the         group consisting of         -   SEQ ID NO:7 and SEQ ID NO:57;         -   SEQ ID NO:8 and SEQ ID NO:58;         -   SEQ ID NO:11 and SEQ ID NO:61;         -   SEQ ID NO:14 and SEQ ID NO:64;         -   SEQ ID NO:16 and SEQ ID NO:66;         -   SEQ ID NO:18 and SEQ ID NO:68;         -   SEQ ID NO:19 and SEQ ID NO:69;         -   SEQ ID NO:25 and SEQ ID NO:75;         -   SEQ ID NO:27 and SEQ ID NO:77;         -   SEQ ID NO:28 and SEQ ID NO:78;         -   SEQ ID NO:29 and SEQ ID NO:79;         -   SEQ ID NO:30 and SEQ ID NO:80; and         -   SEQ ID NO:31 and SEQ ID NO:81.     -   [6] A monoclonal antibody, or antigen-binding fragment thereof,         which comprises a light chain variable region (LCVR) and a heavy         chain variable region (HCVR), wherein said LCVR and said HCVR         comprise, respectively, amino acid sequences selected from the         group consisting of         -   SEQ ID NO:9 and SEQ ID NO:59;         -   SEQ ID NO:10 and SEQ ID NO:60;         -   SEQ ID NO:12 and SEQ ID NO:62;         -   SEQ ID NO:13 and SEQ ID NO:63;         -   SEQ ID NO:15 and SEQ ID NO:65;         -   SEQ ID NO:17 and SEQ ID NO:67;         -   SEQ ID NO:20 and SEQ ID NO:70;         -   SEQ ID NO:21 and SEQ ID NO:71;         -   SEQ ID NO:22 and SEQ ID NO:72;         -   SEQ ID NO:23 and SEQ ID NO:73;         -   SEQ ID NO:24 and SEQ ID NO:74; and         -   SEQ ID NO:26 and SEQ ID NO:76.     -   [7] A monoclonal antibody, or antigen-binding fragment thereof,         that competes with said monoclonal antibody or antigen-binding         fragment thereof of any one of [1]-[6] for binding to CD47.     -   [8] A pharmaceutical composition, comprising said monoclonal         antibody, or antigen-binding fragment thereof, of any one of         [1]-[7], and a pharmaceutically or physiologically acceptable         carrier, diluent, or excipient.     -   [9] A monoclonal antibody, or antigen-binding fragment thereof,         of any one of claims [1]-[7] for use in human therapy or therapy         of companion/pet animals, working animals, sport animals, zoo         animals, or therapy of other valuable animals kept in captivity.     -   [10] The monoclonal antibody, or antigen-binding fragment         thereof, of any one of [1]-[7] for use in treating         ischemia-reperfusion injury, or an autoimmune or inflammatory         disease, in a human or companion/pet animal, working animal,         sport animal, zoo animal, or other valuable animal kept in         captivity.     -   [11] The monoclonal antibody, or antigen-binding fragment         thereof, of [10], which comprises a light chain variable region         (LCVR) and a heavy chain variable region (HCVR), wherein said         LCVR and said HCVR comprise, respectively, amino acid sequences         selected from the group consisting of:         -   SEQ ID NO:7 and SEQ ID NO:57;         -   SEQ ID NO:8 and SEQ ID NO:58;         -   SEQ ID NO:11 and SEQ ID NO:61;         -   SEQ ID NO:14 and SEQ ID NO:64;         -   SEQ ID NO:16 and SEQ ID NO:66;         -   SEQ ID NO:18 and SEQ ID NO:68;         -   SEQ ID NO:19 and SEQ ID NO:69;         -   SEQ ID NO:25 and SEQ ID NO:75;         -   SEQ ID NO:27 and SEQ ID NO:77;         -   SEQ ID NO:28 and SEQ ID NO:78;         -   SEQ ID NO:29 and SEQ ID NO:79;         -   SEQ ID NO:30 and SEQ ID NO:80; and         -   SEQ ID NO:31 and SEQ ID NO:81.     -   [12] The monoclonal antibody, or antigen-binding fragment         thereof, of [10] or [11], wherein said ischemia-reperfusion         injury occurs in organ transplantation, acute kidney injury,         cardiopulmonary bypass surgery, pulmonary hypertension, sickle         cell disease, myocardial infarction, stroke, surgical resections         and reconstructive surgery, reattachment of an appendage or         other body part, skin grafting, or trauma.     -   [13] The monoclonal antibody, or antigen-binding fragment         thereof, of [10] or [11], wherein said autoimmune or         inflammatory disease is selected from the group consisting of         arthritis, multiple sclerosis, psoriasis, Crohn's disease,         inflammatory bowel disease, lupus, Grave's disease and         Hashimoto's thyroiditis, and ankylosing spondylitis.     -   [14] The monoclonal antibody, or antigen-binding fragment         thereof, of any one of [1]-[7] for use in treating a susceptible         cancer.     -   [15] The monoclonal antibody, or antigen-binding fragment         thereof, of [14], which comprises a light chain variable region         (LCVR) and a heavy chain variable region (HCVR), wherein said         LCVR and said HCVR comprise, respectively, amino acid sequences         selected from the group consisting of:         -   SEQ ID NO:9 and SEQ ID NO:59;         -   SEQ ID NO:10 and SEQ ID NO:60;         -   SEQ ID NO:12 and SEQ ID NO:62;         -   SEQ ID NO:13 and SEQ ID NO:63;         -   SEQ ID NO:15 and SEQ ID NO:65;         -   SEQ ID NO:17 and SEQ ID NO:67;         -   SEQ ID NO:20 and SEQ ID NO:70;         -   SEQ ID NO:21 and SEQ ID NO:71;         -   SEQ ID NO:22 and SEQ ID NO:72;         -   SEQ ID NO:23 and SEQ ID NO:73;         -   SEQ ID NO:24 and SEQ ID NO:74; and         -   SEQ ID NO:26 and SEQ ID NO:76.     -   [16] The monoclonal antibody, or antigen binding fragment         thereof, of [14] or [15], which promotes phagocytosis and/or         killing of cells of said susceptible cancer.     -   [17] The monoclonal antibody, or antigen binding fragment         thereof, of any one of [14]-[16], wherein said susceptible         cancer is selected from the group consisting of a leukemia, a         lymphoma, ovarian cancer, breast cancer, endometrial cancer,         colon cancer, rectal cancer, bladder cancer, lung cancer,         bronchial cancer, bone cancer, prostate cancer, pancreatic         cancer, liver and bile duct cancer, esophageal cancer, renal         cancer, thyroid cancer, head and neck cancer, testicular cancer,         glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.     -   [18] The monoclonal antibody, or antigen binding fragment         thereof, of [17], wherein said leukemia is selected from the         group consisting of acute lymphocytic (lymphoblastic) leukemia,         acute myeloid leukemia, chronic lymphocytic leukemia, multiple         myeloma, and chronic myeloid leukemia.     -   [19] Use of said monoclonal antibody, or antigen-binding         fragment thereof, of any one of [1]-[7] to treat         ischemia-reperfusion injury, or an autoimmune or inflammatory         disease, in a human or companion/pet animal, working animal,         sport animal, zoo animal, or other valuable animal kept in         captivity.     -   [20] The use of [19], wherein said monoclonal antibody, or         antigen binding fragment thereof, comprises a light chain         variable region (LCVR) and a heavy chain variable region (HCVR),         wherein said LCVR and said HCVR comprise, respectively, amino         acid sequences selected from the group consisting of:         -   SEQ ID NO:7 and SEQ ID NO:57;         -   SEQ ID NO:8 and SEQ ID NO:58;         -   SEQ ID NO:11 and SEQ ID NO:61;         -   SEQ ID NO:14 and SEQ ID NO:64;         -   SEQ ID NO:16 and SEQ ID NO:66;         -   SEQ ID NO:18 and SEQ ID NO:68;         -   SEQ ID NO:19 and SEQ ID NO:69;         -   SEQ ID NO:25 and SEQ ID NO:75;         -   SEQ ID NO:27 and SEQ ID NO:77;         -   SEQ ID NO:28 and SEQ ID NO:78;         -   SEQ ID NO:29 and SEQ ID NO:79;         -   SEQ ID NO:30 and SEQ ID NO:80; and         -   SEQ ID NO:31 and SEQ ID NO:81.     -   [21] Use of said monoclonal antibody, or antigen-binding         fragment thereof, of any one of [1]-[7] to treat a susceptible         cancer.     -   [22] The use of [21], wherein said monoclonal antibody, or         antigen binding fragment thereof, comprises a light chain         variable region (LCVR) and a heavy chain variable region (HCVR),         wherein said LCVR and said HCVR comprise, respectively, amino         acid sequences selected from the group consisting of:         -   SEQ ID NO:9 and SEQ ID NO:59;         -   SEQ ID NO:10 and SEQ ID NO:60;         -   SEQ ID NO:12 and SEQ ID NO:62;         -   SEQ ID NO:13 and SEQ ID NO:63;         -   SEQ ID NO:15 and SEQ ID NO:65;         -   SEQ ID NO:17 and SEQ ID NO:67;         -   SEQ ID NO:20 and SEQ ID NO:70;         -   SEQ ID NO:21 and SEQ ID NO:71;         -   SEQ ID NO:22 and SEQ ID NO:72;         -   SEQ ID NO:23 and SEQ ID NO:73;         -   SEQ ID NO:24 and SEQ ID NO:74; and         -   SEQ ID NO:26 and SEQ ID NO:76.     -   [23] Use of said monoclonal antibody, or antigen-binding         fragment thereof, of any one of [1]-[7] for the manufacture of a         medicament to treat ischemia-reperfusion injury, or an         autoimmune or inflammatory disease, in a human or companion/pet         animal, working animal, sport animal, zoo animal, or other         valuable animal kept in captivity.     -   [24] The use of [23], wherein said monoclonal antibody, or         antigen binding fragment thereof, comprises a light chain         variable region (LCVR) and a heavy chain variable region (HCVR),         wherein said LCVR and said HCVR comprise, respectively, amino         acid sequences selected from the group consisting of:         -   SEQ ID NO:7 and SEQ ID NO:57;         -   SEQ ID NO:8 and SEQ ID NO:58;         -   SEQ ID NO:11 and SEQ ID NO:61;         -   SEQ ID NO:14 and SEQ ID NO:64;         -   SEQ ID NO:16 and SEQ ID NO:66;         -   SEQ ID NO:18 and SEQ ID NO:68;         -   SEQ ID NO:19 and SEQ ID NO:69;         -   SEQ ID NO:25 and SEQ ID NO:75;         -   SEQ ID NO:27 and SEQ ID NO:77;         -   SEQ ID NO:28 and SEQ ID NO:78;         -   SEQ ID NO:29 and SEQ ID NO:79;         -   SEQ ID NO:30 and SEQ ID NO:80; and         -   SEQ ID NO:31 and SEQ ID NO:81.     -   [25] The use of [23] or [24], wherein said ischemia-reperfusion         injury occurs in organ transplantation, acute kidney injury,         cardiopulmonary bypass surgery, pulmonary hypertension, sickle         cell disease, myocardial infarction, stroke, surgical resections         and reconstructive surgery, reattachment of an appendage or         other body part, skin grafting, and trauma.     -   [26] The use of [23] or [24], wherein said autoimmune or         inflammatory disease is selected from the group consisting of         arthritis, multiple sclerosis, psoriasis, Crohn's disease,         inflammatory bowel disease, lupus, Grave's disease and         Hashimoto's thyroiditis, and ankylosing spondylitis.     -   [27] Use of said monoclonal antibody, or antigen-binding         fragment thereof, of any one of [1]-[7] for the manufacture of a         medicament to treat a susceptible cancer.     -   [28] The use of [27], wherein said monoclonal antibody, or         antigen-binding fragment thereof, comprises a light chain         variable region (LCVR) and a heavy chain variable region (HCVR),         wherein said LCVR and said HCVR comprise, respectively, amino         acid sequences selected from the group consisting of:         -   SEQ ID NO:9 and SEQ ID NO:59;         -   SEQ ID NO:10 and SEQ ID NO:60;         -   SEQ ID NO:12 and SEQ ID NO:62;         -   SEQ ID NO:13 and SEQ ID NO:63;         -   SEQ ID NO:15 and SEQ ID NO:65;         -   SEQ ID NO:17 and SEQ ID NO:67;         -   SEQ ID NO:20 and SEQ ID NO:70;         -   SEQ ID NO:21 and SEQ ID NO:71;         -   SEQ ID NO:22 and SEQ ID NO:72;         -   SEQ ID NO:23 and SEQ ID NO:73;         -   SEQ ID NO:24 and SEQ ID NO:74; and         -   SEQ ID NO:26 and SEQ ID NO:76.     -   [29] A method of treating ischemia or ischemia-reperfusion         injury in a patient in need thereof, comprising administering to         said patient an effective amount of said monoclonal antibody, or         antigen-binding fragment thereof, of any one of [1]-[7], or a         monoclonal antibody, or antigen-binding fragment thereof, that         competes with said monoclonal antibody or antigen-binding         fragment thereof of any one of [1]-[7] for binding to CD47.     -   [30] The method of [29], wherein said patient is about to be         subjected to, or is experiencing, ischemia or         ischemia-reperfusion injury.     -   [31] The method of [29] or [30], wherein said patient is a         human.     -   [32] The method of [29] or [30], wherein said patient is a         companion/pet animal, working animal, sport animal, zoo animal,         or other valuable animal kept in captivity.     -   [33] The method of any one of [29]-[32], wherein said ischemia         occurs because said patient will undergo, or is undergoing, a         surgery selected from the group consisting of integument         surgery, soft tissue surgery, composite tissue surgery, cosmetic         surgery, surgical resections, reconstructive surgery, skin graft         surgery, and limb reattachment surgery.     -   [34] The method of [33], wherein said skin graft is an         autograft.     -   [35] The method of any one of [29]-[32], wherein said ischemia         occurs because said patient will undergo, or is undergoing,         organ transplant surgery.     -   [36] The method of any one of [29]-[32], wherein said         ischemia-reperfusion injury occurs in organ transplantation,         acute kidney injury, cardiopulmonary bypass surgery, pulmonary         hypertension, sickle cell disease, myocardial infarction,         stroke, surgical resection, reconstructive surgery, reattachment         of an appendage or other body part, or skin grafting.     -   [37] The method of any one of [29]-[36], wherein said monoclonal         antibody, antigen-binding fragment thereof, or competing         monoclonal antibody or antigen binding fragment thereof, is         administered before, during, or after said subject undergoes         ischemia or surgery, or a combination of any of these time         periods.     -   [38] The method of any one of [29]-[37], further comprising         administering to said patient an effective amount of a nitric         oxide donor, precursor, or both.     -   [39] The method of [38], wherein said nitric oxide donor or         precursor is selected from the group consisting of NO gas,         isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin,         3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine         (SNAP), Diethylenetriamine/NO (DETACNO), S-nitrosothiols, and         arginine.     -   [40] A method of increasing tissue perfusion in a subject in         need thereof, comprising administering to said subject an         effective amount of a monoclonal antibody, or antigen-binding         fragment thereof, of any one of [1]-[7], or a monoclonal         antibody, or antigen-binding fragment thereof, that competes         with said monoclonal antibody or antigen-binding fragment         thereof of any one of [1]-[7] for binding to CD47.     -   [41] The method of [40], wherein said subject has, or is at risk         of developing, at least one disease or condition selected from         the group consisting of ischemia-reperfusion injury, myocardial         infarction, myocardial ischemia, stroke, cerebral ischemia,         sickle cell anemia, and pulmonary hypertension.     -   [42] The method of [40], wherein said subject has, or is at risk         of developing, at least one disease or condition selected from         the group consisting of hypertension, atherosclerosis,         vasculopathy, ischemia secondary to diabetes, and peripheral         vascular disease.     -   [43] The method of [40], wherein the need for increased tissue         perfusion arises because said subject has had, is having, or         will have, a surgery selected from the group consisting of         integument surgery, soft tissue surgery, composite tissue         surgery, skin graft surgery, resection of a solid organ, and         reattachment or an appendage or other body part.     -   [44] The method of [43], wherein said skin graft is an         autograft.     -   [45] The method of [40], wherein the need for increased tissue         perfusion arises because said subject has had, is having, or         will have, organ transplant surgery.     -   [46] The method of any one of [40]-[45], further comprising         administering to said subject an effective amount of a nitric         oxide donor, precursor, or both.     -   [47] The method of [46], wherein said nitric oxide donor or         precursor is selected from the group consisting of NO gas,         isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin,         3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine         (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and         arginine.     -   [48] A method of transplanting a donor organ from an organ donor         to an organ recipient, comprising any single step, any         combination of steps, or all steps selected from the group         consisting of steps i)-iii):         -   i) administering to said organ donor prior to, during, both             prior to and during, after, or any combination thereof,             donation of said donor organ an effective amount of said             monoclonal antibody or antigen-binding fragment thereof of             any one of [1]-[7], and/or a monoclonal antibody, or             antigen-binding fragment thereof, that competes with said             monoclonal antibody or antigen-binding fragment thereof of             any one of [1]-[7] for binding to CD47;         -   ii) contacting said donor organ prior to, during, both prior             to and during, after, or any combination thereof,             transplantation to said organ recipient, and an effective             amount of said monoclonal antibody or antigen-binding             fragment thereof of any one of [1]-[7], and/or a monoclonal             antibody, or antigen-binding fragment thereof, that competes             with said monoclonal antibody or antigen-binding fragment             thereof of any one of [1]-[7] for binding to CD47; and         -   iii) administering to said organ recipient prior to, during,             both prior to and during, after, or any combination thereof,             transplantation of said donor organ to said organ recipient,             an effective amount of said monoclonal antibody or             antigen-binding fragment thereof of any one of [1]-[7],             and/or a monoclonal antibody, or antigen-binding fragment             thereof, that competes with said monoclonal antibody or             antigen-binding fragment thereof of any one of [1]-[7] for             binding to CD47.     -   [49] The method of claim 48, wherein said monoclonal antibody or         antigen-binding fragment thereof of any one of [1]-[7], or         monoclonal antibody, or antigen-binding fragment thereof, that         competes with said monoclonal antibody or antigen-binding         fragment thereof of any one of [1]-[7] for binding to CD47,         reduces ischemia reperfusion injury in said donor organ.     -   [50] The method of [48] or [49], further comprising         administering to said organ donor, said donor organ, said organ         recipient, or any combination thereof, an effective amount of a         nitric oxide donor, precursor, or both.     -   [51] The method of [50], wherein said nitric oxide donor or         precursor is selected from the group consisting of NO gas,         isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin,         3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine         (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and         arginine.     -   [52] A method of treating an autoimmune or inflammatory disease         in a patient in need thereof, comprising administering to said         patient an effective amount of said monoclonal antibody, or         antigen-binding fragment thereof, of any one of [1]-[7], or a         monoclonal antibody, or antigen-binding fragment thereof, that         competes with said monoclonal antibody or antigen-binding         fragment thereof of any one of [1]-[7] for binding to CD47.     -   [53] The method of [52], wherein said autoimmune or inflammatory         disease is selected from the group consisting of arthritis,         multiple sclerosis, psoriasis, Crohn's disease, inflammatory         bowel disease, lupus, Grave's disease and Hashimoto's         thyroiditis, and ankylosing spondylitis.     -   [54] The method of [52] or [53], wherein said patient is a         human.     -   [55] The method of [52] or [53], wherein said patient is a         companion/pet animal, working animal, sport animal, zoo animal,         or other valuable animal kept in captivity.     -   [56] The method of any one of [52]-[55], further comprising         administering to said patient an effective amount of a nitric         oxide donor, precursor, or both.     -   [57] The method of [56], wherein said nitric oxide donor or         precursor is selected from the group consisting of NO gas,         isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin,         3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine         (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and         arginine.     -   [58] A method of treating a susceptible cancer in a human or         companion/pet animal, working animal, sport animal, zoo animal,         or other valuable animal kept in captivity in need thereof,         comprising administering thereto an effective amount of a         monoclonal antibody or antigen binding fragment thereof of any         one of [1]-[7], or a monoclonal antibody, or antigen-binding         fragment thereof, that competes with said monoclonal antibody or         antigen-binding fragment thereof of any one of [1]-[7] for         binding to CD47, and which exhibits cytotoxic activity.     -   [59] The method of [58], wherein said susceptible cancer is         selected from the group consisting of a leukemia, a lymphoma,         ovarian cancer, breast cancer, endometrial cancer, colon cancer,         rectal cancer, bladder cancer, lung cancer, bronchial cancer,         bone cancer, prostate cancer, pancreatic cancer, liver and bile         duct cancer, esophageal cancer, renal cancer, thyroid cancer,         head and neck cancer, testicular cancer, glioblastoma,         astrocytoma, melanoma, and leiomyosarcoma.     -   [60] The method of [59], wherein said leukemia is selected from         the group consisting of acute lymphocytic (lymphoblastic)         leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,         multiple myeloma, and chronic myeloid leukemia.     -   [61] The method of any one of [58]-[60], wherein said monoclonal         antibody or antigen-binding fragment thereof of any one of         [1]-[7], or said monoclonal antibody, or antigen-binding         fragment thereof, that competes with said monoclonal antibody or         antigen-binding fragment thereof of any one of [1]-[7] for         binding to CD47 and which exhibits cytotoxic activity, increases         phagocytosis of cells of said susceptible cancer.     -   [62] The method of [61], wherein said monoclonal antibody or         antigen-binding fragment thereof of any one of [1]-[7], or said         monoclonal antibody, or antigen-binding fragment thereof, that         competes with said monoclonal antibody or antigen-binding         fragment thereof of any one of [1]-[7] for binding to CD47 which         exhibits cytotoxic activity and increases phagocytosis of cells         of said susceptible cancer inhibits CD47 binding to SIRPalpha.     -   [63] The method of any one of [58] to [62], wherein said         monoclonal antibody or antigen-binding fragment thereof of any         one of [1]-[7], or said monoclonal antibody, or antigen-binding         fragment thereof, that competes with said monoclonal antibody or         antigen-binding fragment thereof of any one of [1]-[7] for         binding to CD47 and which exhibits cytotoxic activity, is         directly toxic to cells of said susceptible cancer.

Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

DEFINITIONS

A full-length antibody as it exists naturally is an immunoglobulin molecule comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

The CDRs are interspersed with regions that are more conserved, termed framework regions (“FR”). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions are in accordance with the well-known Kabat numbering convention.

Light chains are classified as kappa or lambda, and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotope of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.

As used herein, the term “monoclonal antibody” (mAb) as applied to the present antibody compounds refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. mAbs of the present invention preferably exist in a homogeneous or substantially homogeneous population. Complete mAbs contain two heavy chains and two light chains. “Antigen binding fragments” of such monoclonal antibodies include, for example, Fab fragments, Fab′ fragments, F(ab′)₂ fragments, single chain Fv fragments, and one-armed antibodies comprising a light chain and a heavy chain. Monoclonal antibodies and antigen-binding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such technologies, or other technologies known in the art.

“Antibody compounds” refers to mAbs and Fabs, and competing antibodies, disclosed herein. Additional antibody compounds exhibiting similar functional properties according to the present invention can be generated by conventional methods. For example, mice can be immunized with human CD47 or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods disclosed in Examples 3 and 4, below. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15, ISBN 0-87969-314-2.

The phrase “humanized antibodies” refers to monoclonal antibodies and antigen binding fragments thereof in addition to the antibody compounds disclosed herein that have binding and functional properties according to the invention similar to those disclosed herein, and that have framework regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody. “Framework region” or “framework sequence” refers to any one of framework regions 1 to 4. Humanized antibodies and antigen binding fragments encompassed by the present invention include molecules wherein any one or more of framework regions 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human framework regions 1 to 4, is present. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework region 1, 2, and 3, etc., are substantially or fully human. Substantially human frameworks are those that have at least about 80% sequence identity to a known human germline framework sequence. Preferably, the substantially human frameworks have at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to a known human germline framework sequence.

Fully human frameworks are those that are identical to a known human germline framework sequence. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) via their website or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. For example, germline light chain frameworks can be selected from the group consisting of: A11, A17, A18, A19, A20, A27, A30, LI, L1I, L12, L2, L5, L15, L6, L8, O12, O2, and O8, and germline heavy chain framework regions can be selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46, VH3-9, VH3-66, VH3-74, VH4-31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-5I.

Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present invention can be generated using several different methods. In one approach, the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the sequence of the corresponding framework in the parent antibody compound. In the case of frameworks having fewer than 100 amino acid residues, one, two, or three amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful in humanizing mouse antibodies include U.S. Pat. Nos. 4,816,397; 5,225,539; and 5,693,761; computer programs ABMOD and ENCAD as described in Levitt (1983) J. Mol. Biol. 168:595-620; and the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536.

The identification of residues to consider for back-mutation can be carried out as follows.

When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the “acceptor framework”) is replaced by a framework amino acid from a framework of the parent antibody compound (the “donor framework”): (a) the amino acid in the human framework region of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position; (b) the position of the amino acid is immediately adjacent to one of the CDRs; or (c) any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.

When each of the amino acids in the human framework region of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody compound.

Another approach to generating human engineered antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as or better than those of the parent antibody compounds. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.

Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific framework regions in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu et al. (1999) J. Mol. Biol. 294:151-162.

The method described in Example 1 below can also be employed.

Applying the teachings of the present invention, a person skilled in the art can use common techniques, e.g., site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and framework sequences and thereby generate further variable region amino acid sequences derived from the present sequences. Up to all naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to screen these additional variable region amino acid sequences to identify sequences having the indicated in vivo functions. In this way, further sequences suitable for preparing human engineered antibodies and antigen-binding portions thereof in accordance with the present invention can be identified. Preferably, amino acid substitution within the frameworks is restricted to one, two, or three positions within any one or more of the 4 light chain and/or heavy chain framework regions disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, or three positions within any one or more of the 3 light chain and/or heavy chain CDRs. Combinations of the various changes within these framework regions and CDRs described above are also possible.

That the functional properties of the antibody compounds generated by introducing the amino acid modifications discussed above conform to those exhibited by the specific molecules disclosed herein can be confirmed by the methods disclosed below in Examples 3 and 4.

The terms “specifically binds”, “bind specifically”, “specific binding”, and the like as applied to the present antibody compounds refer to the ability of a specific binding agent (such as an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed. A specific binding agent is said specifically to recognize a target molecular species when it can bind specifically to that target.

“Binding affinity” is a term that refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the methods herein that these constants be measured or determined. Rather, affinities as used herein to describe interactions between molecules of the described methods are generally apparent affinities (unless otherwise specified) observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind two other molecules (e.g., two versions or variants of a peptide). The concepts of binding affinity, association constant, and dissociation constant are well known.

The term “epitope” refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

Monoclonal antibodies or antigen-binding fragments thereof that “compete” with the molecules disclosed herein are those that bind human CD47 at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human CD47 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present invention and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such invention antibody compound are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discrete sites on CD47, the labeled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind. For purposes of the present invention, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to CD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pages 567-569, ISBN 0-87969-314-2. Such assays can be made quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor. The capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared. Whether monoclonal antibodies or antigen-binding fragments thereof that compete with antibody compounds of the present invention in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods described in Examples 3 and 4, below.

The term “treating” (or “treat” or “treatment”) means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. The team “treating” and the like refer to a therapeutic intervention that ameliorates a sign, symptom, etc., of a disease or pathological condition after it has begun to develop.

Acute events and chronic conditions can be treated. In an acute event, an antibody or antigen binding fragment thereof is administered at the onset of a symptom, disorder, condition, disease, or procedure, and is discontinued when the acute event ends, or in the case of organ transplantation to the organ, at the time of organ harvest and/or to the transplant recipient at the time of organ transplantation. In contrast, a chronic symptom, disorder, condition, or disease is treated over a more protracted time frame.

The term “effective amount” refers to the amount or dose of an antibody compound of the present invention which, upon single or multiple dose administration to a patient or organ, provides the desired treatment or prevention. Therapeutically effective amounts of the present antibody compounds can comprise an amount in the range of from about 0.1 mg/kg to about 150 mg/kg, more preferably from about 0.1 mg/kg to about 100 mg/kg, and even more preferably from about 0.1 mg/kg to about 50 mg/kg per single dose administered to a harvested organ or to a patient. A therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on a biomarker, such as serum biomarkers of injury of the treated organ, including but not limited to liver, kidney, lung, intestine, pancreas and heart, changes in pulmonary artery pressures, cell surface CD47 expression in tumor or non-tumor tissues, tumor regression, circulating tumor cells or tumor stem cells, etc. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds of the present invention, whether employed alone or in combination with one another, or in combination with another therapeutic agent, or both, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory efficacy are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient.

The antibody compounds of the present invention can be used as medicaments in human and veterinary medicine, administered by a variety of routes. Veterinary applications include the treatment of companion/pet animals, such as cats and dogs; working animals, such as guide or service dogs, and horses; sport animals, such as horses and dogs; zoo animals, such as primates, cats such as lions and tigers, bears, etc.; and other valuable animals kept in captivity.

Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21′ Edition (2005), Lippincott Williams & Wilkins, Philadelphia, Pa., and comprise one or more antibody compounds disclosed herein, and a pharmaceutically or veterinarily acceptable, e.g., physiologically acceptable, carrier, diluent, or excipient.

It should be noted that in all of the therapeutic methods disclosed and claimed herein, the monoclonal antibodies or antigen binding fragments thereof, and monoclonal antibodies or antigen binding fragments thereof that compete with these monoclonal antibodies or antigen binding fragments thereof of the present invention that bind to CD47, can be used alone, or in any appropriate combinations with one another, to achieve the greatest treatment efficacy.

The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, and “tumor” are not mutually exclusive as used herein.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

The term “susceptible cancer” as used herein refers to a cancer, cells of which express CD47 and that are responsive to treatment with an antibody or antigen binding fragment thereof of the present invention. Exemplary susceptible cancers include, but are not limited to, leukemias, including acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, chronic myeloid leukemia, lymphomas, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.

The term “directly toxic” refers to the ability of certain of the humanized antibodies or antigen binding fragments thereof disclosed herein to kill transformed/cancer cells via a cell autonomous mechanism without participation of complement or other cells, including but not limited to, T cells, neutrophils, natural killer cells, macrophages, or dendritic cells.

“Ischemia” refers to a vascular phenomenon in which a decrease in the blood supply to a bodily organ, tissue, or part is caused, for instance, by constriction or obstruction of one or more blood vessels. Ischemia sometimes results from vasoconstriction or thrombosis or embolism. Ischemia can lead to direct ischemic injury, tissue damage due to cell death caused by reduced oxygen supply. Ischemia can occur acutely, as during surgery, or from trauma to tissue incurred in accidents, injuries and war settings, or following harvest of organs intended for subsequent transplantation, for example. It can also occur sub-acutely, as found in atherosclerotic peripheral vascular disease, where progressive narrowing of blood vessels leads to inadequate blood flow to tissues and organs.

When a tissue is subjected to ischemia, a sequence of chemical events is initiated that may ultimately lead to cellular dysfunction and necrosis. If ischemia is ended by the restoration of blood flow, a second series of injurious events ensue, producing additional injury. Thus, whenever there is a transient decrease or interruption of blood flow in a subject, the resultant injury involves two components—the direct injury occurring during the ischemic interval, and the indirect or reperfusion injury that follows.

“Ischemic stroke” can be caused by several different kinds of diseases. The most common problem is narrowing of the arteries in the neck or head. This is most often caused by atherosclerosis, or gradual cholesterol deposition. If the arteries become too narrow, blood cells may collect in them and form blood clots (thrombi). These blood clots can block the artery where they are formed (thrombosis), or can dislodge and become trapped in arteries closer to the brain (embolism). Cerebral stroke can occur when atherosclerotic plaque separates away partially from the vessel wall and occludes the flow of blood through the blood vessel.

“Reperfusion” refers to restoration of blood flow to tissue that is ischemic, due to decrease in blood flow. Reperfusion is a procedure for treating infarction or other ischemia, by enabling viable ischemic tissue to recover, thus limiting further necrosis. However, reperfusion can itself further damage the ischemic tissue, causing reperfusion injury.

In addition to the immediate injury that occurs during deprivation of blood flow, “ischemic/reperfusion injury” involves tissue injury that occurs after blood flow is restored. Current understanding is that much of this injury is caused by chemical products, free radicals, and active biological agents released by the ischemic tissues.

“Nitric oxide donor or precursor” refers to a compound or agent that either delivers NO, or that can be converted to NO through enzymatic or non-enzymatic processes. Examples include, but are not limited to, NO gas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine.

CD47 and Ischemia-Reperfusion Injury (IRI)

Following periods of tissue ischemia, the initiation of blood flow causes damage referred to as “ischemia-reperfusion injury” or IRI. IRI contributes to poor outcomes in many surgical procedures where IRI occurs due to the necessity to stop blood flow for a period of time, in many forms/causes of trauma in which blood flow is interrupted and later restored by therapeutic intervention and in procedures required for organ transplantation, cardio/pulmonary bypass procedures, reattachment of severed body parts, reconstructive and cosmetic surgeries and other situations involving stopping and restarting blood flow. Ischemia itself causes many physiological changes that, by themselves would eventually lead to cell and tissue necrosis and death. Reperfusion poses its own set of damaging events including generation of reactive oxygen species, thrombosis, inflammation and cytokine mediated damage. The pathways that are limited by the TSP1-CD47 system are precisely those that would be of most benefit in combating the damage of IRI. Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosed herein, will provide more robust functioning of these endogenous protective pathways.

The humanized anti-CD47 antibodies, antigen binding fragments thereof, and competing antibodies and antigen binding fragments thereof, of the present invention can be used in the methods disclosed in U.S. Pat. No. 8,236,313, the contents of which are herein incorporated by reference in their entirety.

CD47 and Cancer

CD47 has been identified as a novel therapeutic target in hematologic cancers (Majeti et al. (2009) Cell 138(2):286-99, as well as in solid tumors such as colon, prostate, breast, and brain cancers (Willingham et al. (2012) Proc Natl Acad Sci USA 109(17):6662-7. Many human cancers up-regulate cell surface expression of CD47 and those expressing the highest levels of CD47 are the most aggressive and the most lethal for patients. Increased CD47 expression is thought to protect cancer cells from phagocytic clearance by sending a “don't eat me” signal to macrophages via SIRPalpha, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells (Jaiswal et al. (2009) Cell 138(2):271-851; Chao et al. (2010) Science Translational Medicine 2(63):63ra94). Thus, the increase of CD47 expression by many cancers provides them with a cloak of “selfness” that slows their phagocytic clearance by macrophages and dendritic cells. Anti-CD47 mAbs (CD47mAbs) that block the CD47/SIRPalpha interaction enhance phagocytosis of cancer cells in vitro and contribute to control of tumor burden in published human to mouse xenograft tumor models. However, there are mechanisms by which CD47 mAbs can attack transformed cells that have not yet been exploited in the war on cancer.

Frazier et al. have shown that a particular anti-human CD47mAb (clone 1F7) has a direct, tumor-toxic effect on human T cell leukemias (Manna and Frazier (2003) A. J. Immunol. 170:3544-53) and several breast cancers (Manna and Frazier (2004) A. Cancer Research 64(3):1026-36). Other groups have reported such findings in additional types of leukemia (Uno et al. (2007) Oncol. Rep. 17(5):1189-94; Mateo et al. (1999) Nat. Med. 5:1277-84). MAb 1F7 kills CD47 bearing tumor cells without the action of complement or cell mediated killing by NK cells, T cells or macrophages. Instead, mAb 1F7 acts via a non-apoptotic mechanism that involves a direct CD47-dependent attack on mitochondria, discharging their membrane potential and destroying the ATP-generating capacity of the cell leading to rapid cell death. It is noteworthy that mAb 1F7 does not kill resting leukocytes, which also express CD47, but only those cells that are “activated” by transformation. Thus, normal circulating cells, all of which express CD47, are spared while cancer cells are selectively killed by the tumor-toxic CD47mAb (Manna and Frazier (2003) A. J. Immunol. 170:3544-53). This mechanism can be thought of as a proactive, selective and direct attack on tumor cells in contrast to the passive mechanism of promoting phagocytosis by simply blocking CD47/SIRPalpha binding. Importantly, mAb 1F7 also blocks binding of SIRPalpha to CD47 and thus it can act via two mechanisms: (1) direct tumor cytotoxicity, and (2) promoting phagocytosis of the dead and dying tumor cells. A single mAb that can accomplish both functions may be superior to one that only blocks CD47/SIRPalpha binding. In fact, it has been shown that combining a blocking CD47mAb to promote phagocytosis with the cytotoxic anti-CD20 mAb, rituximab, is more effective than either mAb alone at eradicating human non-Hodgkins lymphoma in a xenograft mouse model (Chao et al. (2010) Cell 142(5):699-713). However, rituximab kills by lysing cancer cells, leading to a harsh side effect profile (Hansel et al. (2010) Nat Rev Drug Discov. 9(4):325-38). In contrast, the tumor-toxic mAb 1F7 does not cause rapid cell lysis, but rather causes display of phosphatidylserine on the cell surface, thus promoting phagocytic clearance by this mechanism as well.

Therapeutic Indications

IRI-Related and Autoimmune/Inflammatory Conditions Administration of a CD47 mAb or antigen binding fragment thereof disclosed herein can be used to treat a number of diseases and conditions in which IRI is a contributing feature, and to treat various autoimmune and inflammatory diseases. These include: organ transplantation in which a mAb or antigen binding fragment thereof of the present invention is administered to the donor prior to organ harvest, to the harvested donor organ, to the organ preservation solution, to the recipient patient, or to any combination thereof; skin grafting; surgical resections or tissue reconstruction in which such mAb or fragment is administered either locally by injection to the affected tissue or parenterally to the patient; reattachment of body parts; treatment of traumatic injury; pulmonary hypertension; sickle cell disease (crisis); myocardial infarction; stroke; surgically-induced ischemia; acute kidney disease/kidney failure; any other condition in which IRI occurs and contributes to the pathogenesis of disease; and autoimmune/inflammatory diseases, including arthritis, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosing spondylitis.

CD47 mAbs and antigen binding fragments thereof of the present invention can also be used to increase tissue perfusion in a subject in need of such treatment. Such subjects can be identified by diagnostic procedures indicating a need for increased tissue perfusion. In addition, the need for increased tissue perfusion may arise because the subject has had, is having, or will have, a surgery selected from integument surgery, soft tissue surgery, composite tissue surgery, skin graft surgery, resection of a solid organ, organ transplant surgery, or reattachment or an appendage or other body part.

Susceptible Cancers

Presently disclosed mAbs effective as cancer therapeutics can be administered to patients, preferably parenterally, with susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, including acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, chronic myeloid leukemia, lymphomas, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.

In certain cases, it may be advantageous to administer the mAb directly to the cancer by injection into the tumor. Since CD47 expression is up-regulated on many cancers, it may also be desirable to use one or more of the disclosed mAbs as imaging and diagnostic agents when labeled with radioactive or other tracers known to those skilled in the art of in vivo imaging of cancers/tumors.

The following examples illustrate various aspects of the present invention, but should not be considered as limiting the invention only to these particularly disclosed embodiments.

EXAMPLE 1 Production of CD47 Antibodies

The humanized antibodies disclosed herein comprise frameworks derived from the human genome. The collection covers the diversity found in the human germ line sequences, yielding functionally expressed antibodies in vivo. The complementarity determining regions (CDRs) in the light and heavy chain variable regions of the target chimeric, non-human antibody VxP037-01LC/VxP037-01HC (SEQ ID NO:7/SEQ ID NO:57) are determined following commonly accepted rules disclosed, for example, in “Protein Sequence and Structure Analysis of Antibody Variable Domains”, In: Antibody Engineering Lab Manual, Eds. S. Duebel and R. Kontermann, Springer-Verlag, Heidelberg (2001)). The CDR fragments are synthesized and combined with pools of frameworks to generate full length variable domains. The humanized variable domains are then combined with a secretion signal and human kappa and human IgG1 constant domains, and cloned into a mammalian expression system (e.g., OptiCHO System, Lifetechnologies, Carlsbad, Calif.) to generate a library of humanized IgG1 variants. An aliquot of the library is sequenced to ensure high diversity and integrity of the reading frames of the individual clones. Aliquots of the humanized variant library are then re-arrayed as single clones into 96 well plates, mini-prepped (e.g., 96 well Miniprep Kit, Qiagen Hilden, Germany), and transfected into CHO cells (Lipofectamine transfection protocol as recommended by Lifetechnologies, Carlsbad, Calif.). Transfected CHO cells are grown in DMEM medium with 10% FBS (both from Lifetechnologies, Carlsbad, Calif.) at 37° C. under 5% CO₂. The humanized variants are expressed as full length IgG 1 molecules, and secreted into the medium.

The cell culture supernatant containing the humanized IgG variants is then screened for binding to the target antigen. In parallel, the concentration of each variant is determined in order to calculate specific activity for each clone. The specific activity of each clone is compared to the specific activity of chimeric clone VxP037-01LC/VxP037-01HC (SEQ ID NO:7/SEQ ID NO:57) expressed on the same plate, and normalized. Top hits from each plate are re-arrayed and re-screened for confirmation. The final candidates are selected by specific activity, functional activity, expression level, and sequence diversity, as well as other criteria, as described below.

EXAMPLE 2 CD47 Antibody CDRs

The amino acid sequences of the light chain and heavy chain variable regions, the complete light and heavy chains, and the respective encoding nucleotide sequences of the foregoing, of the present human engineered antibodies are listed below in the section entitled “Amino Acid and Nucleic Acid Sequences.”

The light chain and heavy chain CDR amino acid sequences are shown in Tables 1 and 2, respectively.

TABLE 1 Light Chain CDRs CDR1 CDR2 CDR3 RSSQSLVHSNGNTYLH KVSYRFS SQNTHVPRT (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3)

TABLE 2 Heavy Chain CDRs CDR1 CDR2 CDR3 GYTFTNYYVF DINPVNGDTNFNEKFKN GGYTMDY (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)

EXAMPLE 3 Binding of Antibodies to CD47 of Different Species

Cross species reactivity of humanized antibodies of the present invention is determined using freshly isolated red blood cells (RBCs), which display CD47 on their surface, from human, mouse, rat, and pig according to the methods disclosed in Kamel et al. (2010) Blood. Transfus. 8(4):260-266.

Supernatants containing secreted antibodies are collected from CHO cells transiently transfected with plasmids encoding antibody clones. Transfected CHO cells are grown in F-12 medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520). Antibody concentration in the supernatants is determined utilizing a quantitative ELISA. ELISA plates are coated with a donkey anti-human FC antibody (Sigma; Catalog #12136) at 10 μg/ml overnight at 4° C. (Promega; Catalog # W4031). Plates are washed with PBS, and then blocked with casein blocking solution (ThermoScientific; Catalog #37532) for 60 minutes at room temperature. Plates are again washed with PBS, tissue culture supernatants are added, and the plates are incubated for 60 minutes at room temperature. Plates are then washed three times with PBS and incubated with peroxidase-conjugated goat anti-human IgG (Jackson Immunoresearch Labs; Catalog #109-035-003) for 60 minutes at room temperature. Plates are washed three times with PBS, and the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine is added (Sigma; Catalog #T4444). Reactions are terminated by the addition of HCl to 0.7N, and absorbance at 450 nM is determined using a Tecan model Infinite M200 plate reader.

RBCs are incubated for 60 minutes on ice with tissue culture supernatants containing the secreted humanized antibodies at a concentration of 10 ng/ml in a solution of phosphate buffered saline, pH 7.2, 2.5 mM EDTA (PBS+E). Cells are then washed with cold PBS+E, and incubated for an additional hour on ice with FITC labeled donkey anti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.; Catalogue #709-096-149) in PBS+E. Cells are then washed with PBS+E, and antibody binding is analyzed using a BD FACSAria Cell Sorter (Becton Dickinson) or a C6 Accuri Flow Cytometer (Becton Dickinson). Antibody binding is quantitated by comparison of mean fluorescence values relative to that of chimeric antibody >VxP037-01LC (SEQ ID NO:7))/>VxP037-01HC (SEQ ID NO:57). The mean fluorescence value for each antibody is divided by the mean fluorescence value for the chimeric antibody.

The results are shown in Table 3, where “Chimera” represents chimeric antibody >VxP037-01LC (SEQ ID NO:7))/>VxP037-01HC (SEQ ID NO:57), Clone 1 represents >pVxK7b-037-hum01-LC (SEQ ID NO:8)/>pVxK7b-037-hum01-HC (SEQ ID NO:58), Clone 2 represents >pVxK7b-037-hum02-LC (SEQ ID NO:9)/>pVxK7b-037-hum02-HC (SEQ ID NO:59), and so on similarly for remaining clones 3-24.

TABLE 3 Binding of Humanized Antibodies to CD47 on the Surface of Red Blood Cells of Different Mammalian Species Clone No. Human Mouse Rat Pig Chimera 1.0 1.0 1.0 1.0 1 1.1 1.7 2.7 1.3 2 1.0 1.2 2.6 1.2 3 0.7 0.9 1.7 0.9 4 0.6 0.6 1.0 0.6 5 1.0 1.0 2.2 1.2 6 0.9 1.2 2.1 1.1 7 0.5 0.4 0.8 0.9 8 0.7 0.7 1.2 0.8 9 1.2 1.4 3.7 1.6 10 1.1 1.2 2.9 1.5 11 0.8 0.7 1.2 1.2 12 0.8 0.6 1.3 1.4 13 1.2 1.3 3.1 1.4 14 1.1 1.5 3.2 1.4 15 1.0 1.3 2.4 1.2 16 0.9 1.0 2.1 1.1 17 0.8 0.9 2.1 1.3 18 1.0 1.3 2.2 1.2 19 0.7 1.0 2.6 1.3 20 1.3 1.5 1.9 1.7 21 1.2 1.2 2.8 1.4 22 1.1 1.2 2.8 1.4 23 1.2 1.4 3.3 1.7 24 0.8 0.7 1.2 1.1

These data demonstrate that all of the humanized CD47 mAb clones disclosed herein bind well to CD47 of a variety of different mammalian species, confirming the useful cross-species reactivity of these antibodies.

EXAMPLE 4 Cell Viability Assay

The purpose of this experiment is to identify antibody clones of the present invention that do, and do not, exhibit cytotoxic activity. For use in cardiovascular indications, including transplantation and other applications related to IRI, the therapeutic mAb should lack cytotoxic activity. In contrast, antibodies useful in the treatment of cancer should exhibit toxicity against transformed/cancer cells. This additional property of selective toxicity to cancer cells is expected to have advantages compared to mAbs that only prevent SIRPalpha binding to CD47.

The method employed is described in Vistica et al. (1991) Cancer Res. 51:2515-2520.

Jurkat JE6.1 cells (ATCC, Manassas, Va.; Catalog # TIB-152) are grown in Iscove's modified Dulbeccco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL, penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1×10⁶ cells/mL For the cell viability assay, cells are plated in 96 well tissue culture plates at a density of 2×10⁴ cells/ml in Iscoves modified Dulbecco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 μg/mL streptomycin (Sigma; #P4222) along with humanized antibodies as disclosed herein at a final concentration of 10 ng/ml, prepared as described above in Example 3. Cells are incubated for 72 hours at 37° C. in an atmosphere of 5% (v/v) CO₂. Cell density is then quantitated using WST1 reagent (Roche Applied Science, Indianapolis, Ind.; Catalog #05015944001) according to the manufacturer's instructions. The effect of the antibodies on cell growth is quantitated by comparison to growth of cells containing no added antibody (PBS; average percent killing=0).

The results are shown in table 4. The values in the table represent the mean of 3 separate experiments. “Chimera” and clone numbers are as described above in Example 3. 1F7 is the anti-human CD47mAb, discussed above, that has a direct, tumor-toxic effect on human T cell leukemias (Manna and Frazier (2003) A. J. Immunol. 170:3544-53) and several breast cancers (Manna and Frazier (2004) A. Cancer Research 64(3):1026-36).

TABLE 4 Cytotoxicity of Humanized CD47mAbs on Transformed Human T Cells, Jurkat JE6.1 Clone No. Average % Killing % of 1F7 Cytotoxic Chimera 4.3 19 1 −3.3 −14 2 9.8 43 Yes 3 8.6 38 Yes 4 6.8 30 5 11.8 52 Yes 6 14 61 Yes 7 1.8 8 8 10.6 46 Yes 9 1 4 10 7.4 32 Yes 11 −7.2 −32 12 −6.9 −30 13 17.8 78 Yes 14 16.5 72 Yes 15 8.1 36 Yes 16 8.7 38 Yes 17 12.4 54 Yes 18 5.4 23 19 9.6 42 Yes 20 3.1 14 21 4.5 20 22 −0.7 −3 23 4.8 21 24 −13.1 −57 1F7 22.9 100 Yes

These data demonstrate that the majority of the present humanized antibody clones are not significantly cytotoxic toward Jurkat T cells. However, certain of the clones have significant cytotoxicity, similar to previously identified mouse anti-human CD47mAb 1F7 (Manna and Frazier, J. Immunol. (2003) 170(7):3544-53.

The following clones, indicated in Table 2 with a “Yes”, are considered to be cytotoxic: 2, 3, 5, 6, 8, 10, 13, 14, 15, 16, 17, and 19.

The following clones are considered to be non-toxic: 1, 4, 7, 9, 11, 12, 18, 20, 21, 22, 23, and 24.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Amino Acid and Nucleic Acid Sequences Light Chain Variable Region Amino Acid Sequences >VxP037-01LC: Underlined amino acid sequences represent CDRs (SEQ ID NO: 7) DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPRTFGQG >pVxK7b-037-hum01-LC (SEQ ID NO: 8) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum02-LC (SEQ ID NO: 9) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVPRTFGQG >pVxK7b-037-hum03-LC (SEQ ID NO: 10) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum04-LC (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVPRTFGQG >pVxK7b-037-hum05-LC (SEQ ID NO: 12) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum06-LC (SEQ ID NO: 13) DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVPRTFGQG >pVxK7b-037-hum07-LC (SEQ ID NO: 14) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum08-LC (SEQ ID NO: 15) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum09-LC (SEQ ID NO: 16) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum10-LC (SEQ ID NO: 17) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum11-LC (SEQ ID NO: 18) EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVPRTFGQG >pVxK7b-037-hum12-LC (SEQ ID NO: 19) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum13-LC (SEQ ID NO: 20) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum14-LC (SEQ ID NO: 21) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum15-LC (SEQ ID NO: 22) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVPRTFGQG >pVxK7b-037-hum16-LC (SEQ ID NO: 23) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum17-LC (SEQ ID NO: 24) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum18-LC (SEQ ID NO: 25) EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVPRTFGQG >pVxK7b-037-hum19-LC (SEQ ID NO: 26) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum20-LC (SEQ ID NO: 27) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum21-LC (SEQ ID NO: 28) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum22-LC (SEQ ID NO: 29) EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVPRTFGQG >pVxK7b-037-hum23-LC (SEQ ID NO: 30) DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPKLLIYKVSYRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQG >pVxK7b-037-hum24-LC (SEQ ID NO: 31) AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPRLLIYKVSYRF SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVPRTFGQG Light Chain Variable Region Nucleic Acid Sequences >VxP037-01LC (SEQ ID NO: 32) GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATC AGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGAG >pVxK7b-037-hum01-LC (SEQ ID NO: 33) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum02-LC (SEQ ID NO: 34) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATC AGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum03-LC (SEQ ID NO: 35) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum04-LC (SEQ ID NO: 36) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATC AGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum05-LC (SEQ ID NO: 37) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum06-LC (SEQ ID NO: 38) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATC AGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum07-LC (SEQ ID NO: 39) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum08-LC (SEQ ID NO: 40) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum09-LC (SEQ ID NO: 41) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum10-LC (SEQ ID NO: 42) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum11-LC (SEQ ID NO: 43) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATC AGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum12-LC (SEQ ID NO: 44) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum13-LC (SEQ ID NO: 45) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum14-LC (SEQ ID NO: 46) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum15-LC (SEQ ID NO: 47) GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATC AGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum16-LC (SEQ ID NO: 48) GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum17-LC (SEQ ID NO: 49) GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum18-LC (SEQ ID NO: 50) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATC AGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum19-LC (SEQ ID NO: 51) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum20-LC (SEQ ID NO: 52) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum21-LC (SEQ ID NO: 53) GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum22-LC (SEQ ID NO: 54) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATC AGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum23-LC (SEQ ID NO: 55) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG >pVxK7b-037-hum24-LC (SEQ ID NO: 56) GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAAAGTTTCCTACCGATTT TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATC AGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGG Heavy Chain Variable Region Amino Acid Sequences >VxP037-01HC (SEQ ID NO: 57) EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGDINPVNGDTNF NEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQG >pVxK7b-037-hum01-HC (SEQ ID NO: 58) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum02-HC (SEQ ID NO: 59) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum03-HC (SEQ ID NO: 60) EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum04-HC (SEQ ID NO: 61) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSDINPVNGDTNF NEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum05-HC (SEQ ID NO: 62) QVQLQESGPGINKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSDINPVNGDTNF NEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum06-HC (SEQ ID NO: 63) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum07-HC (SEQ ID NO: 64) QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum08-HC (SEQ ID NO: 65) QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNF NEKFKNRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum09-HC (SEQ ID NO: 66) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum10-HC (SEQ ID NO: 67) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMINMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum11-HC (SEQ ID NO: 68) QVQLQESGPGINKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum12-HC (SEQ ID NO: 69) QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum13-HC (SEQ ID NO: 70) EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum14-HC (SEQ ID NO: 71) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum15-HC (SEQ ID NO: 72) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum16-HC (SEQ ID NO: 73) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGGYTMDYWGQG >pVxK7b-037-hum17-HC (SEQ ID NO: 74) EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYYVFWIRQPPGKGLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum18-HC (SEQ ID NO: 75) EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum19-HC (SEQ ID NO: 76) EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum20-HC (SEQ ID NO: 77) QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWVRQAPGQGLEWMGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG >pVxK7b-037-hum21-HC (SEQ ID NO: 78) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum22-HC (SEQ ID NO: 79) QVQLQESGPGLVKPSQTLSLICTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum23-HC (SEQ ID NO: 80) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGGYTMDYWGQG >pVxK7b-037-hum24-HC (SEQ ID NO: 81) QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGDINPVNGDTNF NEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYTMDYWGQG Heavy Chain Variable Region Nucleic Acid Sequences >VxP037-01HC (SEQ ID NO: 82) GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAATGAAGTTG TCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGAAACAGAGG CCTGGACAAGGCCTTGAGTGGATTGGAGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAAGGCCACACTGACTGTAGACAAGTCCTCCACCACAACATAC TTGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGT TATACTATGGACTACTGGGGTCAAGGA >pVxK7b-037-hum01-HC (SEQ ID NO: 83) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum02-HC (SEQ ID NO: 84) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum03-HC (SEQ ID NO: 85) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATC TCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum04-HC (SEQ ID NO: 86) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTCAGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCC CTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum05-HC (SEQ ID NO: 87) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTCAGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCC CTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum06-HC (SEQ ID NO: 88) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum07-HC (SEQ ID NO: 89) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATC TCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum08-HC (SEQ ID NO: 90) CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTG ACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCC CCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum09-HC (SEQ ID NO: 91) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum10-HC (SEQ ID NO: 92) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum11-HC (SEQ ID NO: 93) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATC TCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum12-HC (SEQ ID NO: 94) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATC TCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum13-HC (SEQ ID NO: 95) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATC TCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCC CCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum14-HC (SEQ ID NO: 96) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum15-HC (SEQ ID NO: 97) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum16-HC (SEQ ID NO: 98) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA  >pVxK7b-037-hum17-HC (SEQ ID NO: 99) GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATC TCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCCGCCAGCCC CCAGGGAAGGGGCTGGAGTGGATTGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum18-HC (SEQ ID NO: 100) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATC TCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCC CCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum19-HC (SEQ ID NO: 101) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATC TCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGATCAGGCAGTCC CCATCGAGAGGCCTTGAGTGGCTGGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum20-HC (SEQ ID NO: 102) CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTG ACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum21-HC (SEQ ID NO: 103) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum22-HC (SEQ ID NO: 104) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum23-HC (SEQ ID NO: 105) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTC CTTACAATGACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum24-HC (SEQ ID NO: 106) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTACAGTGAAAATC TCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGCGACAGGCT CGTGGACAACGCCTTGAGTGGATAGGTGACATTAATCCTGTCAATGGTGATACTAACTTC AATGAGAAATTCAAGAACAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT TATACTATGGACTACTGGGGCCAGGGA

Complete Light Chain Amino Acid Sequences

>VxP037-01-LC-Pro represents the full length light chain variable domain+constant domain amino acid sequence. The underlined amino acid sequence=framework 4+the constant domain. All the humanized light chain sequences contain the same constant domain as VxP037-01-LC-Pro. However, this is not shown in the remaining humanized light chain amino acid sequences.

>VxP037-01-LC-Pro (SEQ ID NO: 107) DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSYR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPRTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Complete Light Chain Nucleic Acid Sequences

The underlined nucleic acid sequence encodes the underlined protein sequence in >VxP037-01-LC-Pro, above.

>Vx-P037-01-LC-DNA (SEQ ID NO: 108) GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCT CGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGC ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCC TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGTTGA

Complete Heavy Chain Amino Acid Sequences

>VxP037-01-HC-Pro represents the full length heavy chain variable domain+constant domain amino acid sequence. The underlined amino acid sequence=framework 4+the constant domain. All the humanized heavy chain sequences contain the same constant domain as >VxP037-01-HC-Pro. However, this is not shown in the remaining humanized heavy chain amino acid sequences.

>VxP037-01-HC-Pro (SEQ ID NO: 109) EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGDINPVNGDT NFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Complete Heavy Chain Nucleic Acid Sequences

The underlined nucleic acid sequence encodes the underlined protein sequence in >VxP037-01-HC-Pro, above.

>VxP037-01-HC-DNA (SEQ ID NO: 110) GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAATGAAGTTGTC CTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATGTATTCTGGGTGAAACAGAGGCCTG GACAAGGCCTTGAGTGGATTGGAGACATTAATCCTGTCAATGGTGATACTAACTTCAATGAG AAATTCAAGAACAAGGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACT CAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGTTATACTATGG ACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGCCTCCACCAAGGGCCCATCGGTC TTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA ACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCAGCAACAAAGCCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 

1-63. (canceled)
 64. A monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human, rat, mouse, and pig CD47.
 65. The monoclonal antibody, or antigen-binding fragment thereof, of claim 64, which is chimeric or humanized.
 66. The monoclonal antibody, or antigen-binding fragment thereof, of claim 64, which comprises three light chain complementarity determining regions (LCDRs 1-3) and three heavy chain complementarity determining regions (HCDRs 1-3), wherein: LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:1) LCDR 2 comprises the amino acid sequence KVSYRFS (SEQ ID NO:2); and LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID NO:3); HCDR 1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID NO:4); HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN (SEQ ID NO:5); and HCDR 3 comprises the amino acid sequence GGYTMDY(SEQ ID NO:6).
 67. The monoclonal antibody, or antigen-binding fragment thereof, of claim 64, which comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR and said HCVR comprise, respectively, amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:57; SEQ ID NO:8 and SEQ ID NO:58; SEQ ID NO:9 and SEQ ID NO:59; SEQ ID NO:10 and SEQ ID NO:60; SEQ ID NO:11 and SEQ ID NO:61; SEQ ID NO:12 and SEQ ID NO:62; SEQ ID NO:13 and SEQ ID NO:63; SEQ ID NO:14 and SEQ ID NO:64; SEQ ID NO:15 and SEQ ID NO:65; SEQ ID NO:16 and SEQ ID NO:66; SEQ ID NO:17 and SEQ ID NO:67; SEQ ID NO:18 and SEQ ID NO:68; SEQ ID NO:19 and SEQ ID NO:69; SEQ ID NO:20 and SEQ ID NO:70; SEQ ID NO:21 and SEQ ID NO:71; SEQ ID NO:22 and SEQ ID NO:72; SEQ ID NO:23 and SEQ ID NO:73; SEQ ID NO:24 and SEQ ID NO:74; SEQ ID NO:25 and SEQ ID NO:75; SEQ ID NO:26 and SEQ ID NO:76; SEQ ID NO:27 and SEQ ID NO:77; SEQ ID NO:28 and SEQ ID NO:78; SEQ ID NO:29 and SEQ ID NO:79; SEQ ID NO:30 and SEQ ID NO:80; and SEQ ID NO:31 and SEQ ID NO:81.
 68. A monoclonal antibody, or antigen-binding fragment thereof, that competes with said monoclonal antibody, or antigen-binding fragment thereof, of claim 64 for binding to CD47.
 69. A pharmaceutical composition, comprising said monoclonal antibody, or antigen-binding fragment thereof, of claim 64, and a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.
 70. A method of treating a susceptible cancer in a human or companion/pet animal, working animal, sport animal, zoo animal, or other valuable animal kept in captivity in need thereof, comprising administering thereto an effective amount of a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human, rat, mouse, and pig CD47, and which exhibits cytotoxic activity.
 71. The method of claim 70, wherein said monoclonal antibody, or antigen-binding fragment thereof, is chimeric or humanized.
 72. The method of claim 70, wherein said monoclonal antibody, or antigen-binding fragment thereof, comprises three light chain complementarity determining regions (LCDRs 1-3) and three heavy chain complementarity determining regions (HCDRs 1-3), wherein: LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:1) LCDR 2 comprises the amino acid sequence KVSYRFS (SEQ ID NO:2); and LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID NO:3); HCDR1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID NO:4); HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN (SEQ ID NO:5); and HCDR 3 comprises the amino acid sequence GGYTMDY(SEQ ID NO:6).
 73. The method of claim 70, wherein said monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR and said HCVR comprise, respectively, amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:57; SEQ ID NO:8 and SEQ ID NO:58; SEQ ID NO:9 and SEQ ID NO:59; SEQ ID NO:10 and SEQ ID NO:60; SEQ ID NO:11 and SEQ ID NO:61; SEQ ID NO:12 and SEQ ID NO:62; SEQ ID NO:13 and SEQ ID NO:63; SEQ ID NO:14 and SEQ ID NO:64; SEQ ID NO:15 and SEQ ID NO:65; SEQ ID NO:16 and SEQ ID NO:66; SEQ ID NO:17 and SEQ ID NO:67; SEQ ID NO:18 and SEQ ID NO:68; SEQ ID NO:19 and SEQ ID NO:69; SEQ ID NO:20 and SEQ ID NO:70; SEQ ID NO:21 and SEQ ID NO:71; SEQ ID NO:22 and SEQ ID NO:72; SEQ ID NO:23 and SEQ ID NO:73; SEQ ID NO:24 and SEQ ID NO:74; SEQ ID NO:25 and SEQ ID NO:75; SEQ ID NO:26 and SEQ ID NO:76; SEQ ID NO:27 and SEQ ID NO:77; SEQ ID NO:28 and SEQ ID NO:78; SEQ ID NO:29 and SEQ ID NO:79; SEQ ID NO:30 and SEQ ID NO:80; and SEQ ID NO:31 and SEQ ID NO:81.
 74. The method of claim 70, wherein said susceptible cancer is selected from the group consisting of a leukemia, a lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, and leiomyosarcoma.
 75. The method of claim 74, wherein said leukemia is selected from the group consisting of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, and chronic myeloid leukemia.
 76. The method of claim 70, wherein said monoclonal antibody, or antigen-binding fragment thereof, which exhibits cytotoxic activity, increases phagocytosis of cells of said susceptible cancer.
 77. The method of claim 76, wherein said monoclonal antibody, or antigen-binding fragment thereof, which exhibits cytotoxic activity and increases phagocytosis of cells of said susceptible cancer, inhibits CD47 binding to SIRPalpha.
 78. The method of claim 70, wherein said monoclonal antibody, or antigen-binding fragment thereof, which exhibits cytotoxic activity, is directly toxic to cells of said susceptible cancer.
 79. A method of treating ischemia or ischemia-reperfusion injury in a patient in need thereof, comprising administering to said patient an effective amount of a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds human, rat, mouse, and pig CD47.
 80. The method of claim 79, wherein said monoclonal antibody, or antigen-binding fragment thereof, comprises three light chain complementarity determining regions (LCDRs 1-3) and three heavy chain complementarity determining regions (HCDRs 1-3), wherein: LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:1) LCDR 2 comprises the amino acid sequence KVSYRFS (SEQ ID NO:2); and LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID NO:3); HCDR1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID NO:4); HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN (SEQ ID NO:5); and HCDR 3 comprises the amino acid sequence GGYTMDY(SEQ ID NO:6).
 81. The method of claim 79, wherein said monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR and said HCVR comprise, respectively, amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:57; SEQ ID NO:8 and SEQ ID NO:58; SEQ ID NO:9 and SEQ ID NO:59; SEQ ID NO:10 and SEQ ID NO:60; SEQ ID NO:11 and SEQ ID NO:61; SEQ ID NO:12 and SEQ ID NO:62; SEQ ID NO:13 and SEQ ID NO:63; SEQ ID NO:14 and SEQ ID NO:64; SEQ ID NO:15 and SEQ ID NO:65; SEQ ID NO:16 and SEQ ID NO:66; SEQ ID NO:17 and SEQ ID NO:67; SEQ ID NO:18 and SEQ ID NO:68; SEQ ID NO:19 and SEQ ID NO:69; SEQ ID NO:20 and SEQ ID NO:70; SEQ ID NO:21 and SEQ ID NO:71; SEQ ID NO:22 and SEQ ID NO:72; SEQ ID NO:23 and SEQ ID NO:73; SEQ ID NO:24 and SEQ ID NO:74; SEQ ID NO:25 and SEQ ID NO:75; SEQ ID NO:26 and SEQ ID NO:76; SEQ ID NO:27 and SEQ ID NO:77; SEQ ID NO:28 and SEQ ID NO:78; SEQ ID NO:29 and SEQ ID NO:79; SEQ ID NO:30 and SEQ ID NO:80; and SEQ ID NO:31 and SEQ ID NO:81.
 82. The method of claim 79, further comprising administering to said patient an effective amount of a nitric oxide donor, precursor, or both.
 83. The method of claim 82, wherein said nitric oxide donor or precursor is selected from the group consisting of NO gas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, and arginine. 